Fibrosis is the final pathologic result common to several chronic inflammatory diseases. Although collagen deposition is an unavoidable and generally reversible step of wound healing, normal tissue repair can evolve into an irreversible and progressive fibrotic response if tissue damage is serious or repeated or if healing response gets out of control. Fibrosis is defined as an excessive accumulation of fibrous connective tissue, i.e. of components of the extracellular matrix (ECM) such as collagen and fibronectin around the inflammatory or damaged tissue, which can lead to permanent scars, organ dysfunction and eventually to death, as has been seen in the late stage of hepatic and renal disease, in idiopathic pulmonary fibrosis (IPF) and in heart failure. Fibrosis is also a pathologic feature of several chronic autoimmune diseases including dermatosclerosis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, myelofibrosis and systemic lupus erythematosus. Moreover, fibrosis affects tumor invasion and metastases, chronic rejection and the pathogenesis of several progressive myopathies (Wynn et al., Nat. Med 2012; 18(7): 1028-1040).
Despite the fact that fibrogenesis is recognized as one of the main causes of morbidity and mortality in most chronic inflammatory and autoimmune diseases, few treatments are available specifically addressing fibrosis pathogenesis.
Fibrotic phenomena and/or alterations of the extracellular matrix also play an important role in tumor diseases. The local microenvironment, or niche, of a tumor cell is indeed a key to cancer development. An important component of this niche is the extracellular matrix (ECM), a complex network of macromolecules comprising collagen and fibronectin and having peculiar physical, biochemical and biomechanical properties. Though being closely controlled during embryonic development and organ homeostasis, EMC is remodeled in diseases like cancer. An abnormal ECM affects transformation promoting cancer progression and metastasis. As a matter of fact, ECM abnormalities affect the behavior of stromal, endothelial and immune system cells creating an inflammatory and pro-tumorigenic environment (Pengfei et al., J. Cell Biol. 2012; 196(4): 395-406; Arendt et al., Semin Cell Dev Biol. 2010; 21(1): 11-18; Martinez-Outschoorn et al., Int J Biochem Cell Biol. 2011; 43(7): 1045-1051; Li et al., Curr Pharm Des. 2012; 18(17): 2404-2415; Sherman et al., Cancer Prey Res (Phila). 2012; 5(1): 3-1; Giatromanolaki et al., Cancer Biology & Therapy 2000; 6(5): 639-645). Therefore, new therapeutic agents should aim at preventing both the progressive ECM deregulation and the activation of stromal cells by acting upon the local tumor microenvironment.
Finally, it is known that the presence of a strong fibrotic component, produced by microenvironment cells and by tumor cells themselves subjected to epithelial mesenchymal transition, can affect the response to chemotherapeutic therapy. As a matter of fact, the content and structural organization of collagens, by increasing the density of the matrix and the pressure of the interstitial fluid, can negatively affect the accessibility of drugs to tumor (Egebla et al., Curr Opin Cell Biol 2010. 22: 697-706). Therefore, the administration in combination with an antifibrotic agent can be useful in enhancing the activity of chemotherapeutic drugs, enabling to administer to the patient a smaller drug dose and as a result to reduce the adverse effects thereof, or anyhow to obtain a higher therapeutic effectiveness. The occurrence of fibrotic phenomena is also an adverse effect of radiotherapy and of some chemotherapeutic drugs, such as e.g. bleomycin, fludarabine and methotrexate. Therefore, the combined administration of radiotherapy or of these chemotherapeutic drugs with antifibrotic agents can inhibit fibrosis-related tissue damage and thus reduce the adverse effects associated to the administration thereof.
The gene for JMJD6 was cloned in 2000 and at first the protein was incorrectly classified as a transmembrane protein. Based on the characteristics of the residues present on the putative extracellular domain of the protein and on in-vitro data, it was assumed at first that this protein was located on the surface of macrophages, where it was able to bind phosphatidylserine, thus regulating the phagocytosis of apoptotic cells induced by the exposition of this specific phospholipid on the surface thereof. Based on these experimental evidence, the protein was classified at first as a phosphatidylserine receptor (Fadok et al., Nature 2000; 405: 85-90).
However, these initial assumptions were discredited by following studies, which conversely pointed out that JMJD6 is not a transmembrane protein but it is only located in the nucleus (Cikala et al., BMC Cell Biol 2004; 5:26; Cui et al., Experimental Cell Research 2004; 293:154-163.
Moreover, also its role in the removal of apoptotic cells was confuted (Bose et al., J Biol 2004; 3:15). In Zakharova et al., (J Cell Physiol 2009; 221: 84-91) it is stated that JMJD6 is expressed on the surface of immature phagocytes, but it is translocated into the nucleus as a result of cell differentiation.
The protein was recognized at the same time as a member of the family of proteins containing the JmjC domain and renamed JMJD6 by the International Committee for Standardized Genetic Nomenclature in Mice (ICSGNM).
In affinity with other members of this family, JMJD6 was attributed a demethylation activity on arginine residues of histone 3 and 4 (Chang et al., Science 2007; 318: 444-447). However, the presence of this enzymatic activity is currently under discussion since the observation could not be reproduced in following studies (Hahn et al., PLos One 2010; 5(10): 13769 and Webby et al., Science 2009; 325: 90-93). Webby et al. assumes that this inconsistency can be due to the fact that the ability to catalyze demethylation of arginine residues is very weak and therefore not always detectable.
More recently, it was proved that the main enzymatic activity JMJD6 is on the contrary the hydroxylation of lysine residues. Through this activity on some splicing factors, the protein plays an important role in the regulation of alternative splicing and thus in gene regulation (Webby et al., Science 2009; 325: 90-93; Hahn et al., BMC Genomics 2008; 9:293 and Hahn et al., pLos One 2010; 5(10): e13769); Unoki M et al., J Biol Chem. 2013; 288(9):6053-62. Poulard C et al., PLoS One 2014; 9(2):e87982; Wang F et al., PLoS Biol. 2014; 12(3):e1001819; Heim A et al., Nucleic Acids Res. 2014; 42 (12): 7833-50.
More recent literature agrees on considering JMJD6 as a nuclear protein with functions related to the regulation of gene expression.
Koninger et al., Annals of Surgery 2005; 241 (1): 144-151; Vandivier et al., Journal Clinic Invest 2002; 109(5): 661-670, and WO01/0066785 assume that the stimulation of phosphatidylserine receptor (first name of JMJD6) can induce the secretion of TGF-beta, a molecule whose profibrotic activity is known. However, this assumption arises from experimental observations which attributed a role to the phosphatidylserine receptor (PSR-1) in the phagocytosis of apoptotic cells, which role was then rejected. However, although recent literature no longer attributes this function to JMJD6, PSR-1 has been recently reported to recognize phosphatidylserine on the surface of apoptotic cells, in C. Elegans (Yang H. et al. Nat Commun 2015; 6: 5717).
WO2009141609 describes modulators of JMJD6 enzymatic activity for the treatment of diseases related to an abnormal RNA splicing, such as e.g. tumor diseases. WO2010101528 describes JMJD6 as a diagnostic and prognostic bio-marker of breast cancer, which can distinguish between the tumor at an initial stage and a metastatic and/or advanced tumor. In this context, the document assumes the use of JMJD6 antagonists, including antibodies, in the prevention and reduction of tumor metastases. Neither does the document provide details about the antibody characteristics required to obtain a desired antagonist activity, nor a role of JMJD6 outside the cell, in the stroma, is assumed, nor a direct involvement thereof in fibrosis is assumed. Lastly, Wang F et al., PLoS Biol. 2014; 12(3):e1001819 describes that depletion of JMJD6 by two specific siRNA represses p53-dependent colon cell proliferation and tumorigenesis, a finding that neither makes reference to the protein outside the cells and its interaction with collagen on other ECM proteins nor to fibrosis.